Selective Targeting Agents for Mitochondria

ABSTRACT

The present invention provides a composition and related methods for delivering cargo to a mitochondria which includes (a) a membrane active peptidyl fragment having a high affinity with the mitochondria and (b) cargo. The cargo may be selected from a wide variety of desired cargos which are to be delivered to the mitochondria for a specific purpose. Compositions and methods are disclosed for treating an illness that is caused or associated with cellular damage or dysfunction which is caused by excessive mitochondrial production of reaction oxygen species (ROS). Compositions which act as mitochondria-selective targeting agents using the structural signaling of the β-turn recognizable by cells as mitochondria) targeting sequences are discussed. Mitochondria and cell death by way of apoptosis is inhibited as a result of the ROS-scavenging activity, thereby increasing the survival rate of the patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/058,461, filed Oct. 21, 2013, now U.S. Pat. No. 9,006,186, issuedApr. 14, 2015, which is a continuation of U.S. patent application Ser.No. 12/750,891, filed Mar. 31, 2010, now abandoned, which is acontinuation of U.S. patent application Ser. No. 11/565,779, filed Dec.1, 2006, now U.S. Pat. No. 7,718,603, issued May 18, 2010, which is acontinuation-in-part of U.S. patent application Ser. No. 11/465,524,filed Aug. 18, 2006, now abandoned, which was a continuation-in-part ofU.S. patent application Ser. No. 11/465,162, filed Aug. 17, 2006, nowU.S. Pat. No. 7,528,174, issued May 5, 2009, which in turn claimed thebenefit of U.S. Provisional Application No. 60/757,044 entitled“Selective Targeting Agents for Mitochondria” filed on Jan. 6, 2006,each of which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.W81XWH-05-2-0026, awarded by DARPA, and Grant No. GM067082, awarded bythe National Institutes of Health. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods for providingmitochondria-selective targeting agents bonded or linked to desiredcargo, such as radical scavenging agents. In another embodiment, thecargo transported by mitochondrial-selective targeting agents mayinclude an inhibitor of nitric oxide synthase (“NOS”) enzyme activity.Some embodiments employ membrane active peptidyl fragments having a highaffinity with the mitochondria and the cargo. Some embodiments focus oncompositions and methods of TEMPO conjugates, particularly syntheticGramicidin S-peptidyl TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl)conjugates.

2. Description of the Prior Art

Cells typically undergo some degree of oxidative stress by way ofgenerating reactive oxygen species (“ROS”) and reactive nitrogen species(“RNS”). Specifically, the cellular respiration pathway generates ROSand RNS within the mitochondrial membrane of the cell, see Kelso et al.,Selective Targeting of a Redox-active Ubiquinone to Mitochondria withinCells: Antioxidant and Antiapoptotic Properties, J. BIOL. CHEM. 276:4588(2001). Reactive oxygen species include free radicals, reactive anionscontaining oxygen atoms, and molecules containing oxygen atoms that caneither produce free radicals or are chemically activated by them.Specific examples include superoxide anion, hydroxyl radical, andhydroperoxides.

Naturally occurring enzymes, such as superoxide dismutase (“SOD”) andcatalase salvage ROS and RNS radicals to allow normal metabolic activityto occur.

Significant deviations from cell homeostasis, such as hemorrhagic shock,lead to an oxidative stress state, thereby causing “electron leakage”from the mitochondrial membrane. Said “electron leakage” produces anexcess amount of ROS for which the cell's natural antioxidants cannotcompensate. Specifically, SOD cannot accommodate the excess productionof ROS associated with hemorrhagic shock which ultimately leads topremature mitochondria dysfunction and cell death via apoptosis, seeKentner et al., Early Antioxidant Therapy with TEMPOL during HemorrhagicShock Increases Survival in Rats, J. OF TRAUMA® INJURY, INFECTION, ANDCRITICAL CARE, 968 (2002).

Cardiolipin (“CL”) is an anionic phospholipid exclusively found in theinner mitochondrial membrane of eukaryotic cells, see Iverson, S. L. andS. Orrenius, The cardiolipin-cytochrome c interaction and themitochondria) regulation of apoptosis, ARCH. BIOCHEM. 423:37-46 (2003).

Under normal conditions, the pro-apoptotic protein cytochrome c isanchored to the mitochondrial inner membrane by binding with CL, seeTuominen, E. K. J., et al. Phospholipid cytochrome c interaction:evidence for the extended lipid anchorage, J. BIOL. CHEM., 277:8822-8826(2002). The acyl moieties of CL are susceptible to peroxidation byreactive oxygen species. When ROS are generated within mitochondria inexcess quantities, cytochrome C bound to CL can function as an oxidaseand induces extensive peroxidation of CL in the mitochondrial membrane,see Kagan, V. E. et al., Cytochrome c acts as a cardiolipin oxygenaserequired, for release of proapoptotic, factors, NATURE CHEM. BIOL.1:223-232 (2005); also Kagan, V. E. et al., Oxidative lipidomics ofapoptosis: redox catalytic interactions of cytochrome c with cardiolipinand phosphatidylserine, FREE RAH. BIOL. MED. 37:1963-1985 (2005).

The peroxidation of the CL weakens the binding between the CL andcytochrome C, see Shidoji, Y. et al., Loss of molecular interactionbetween cytochrome C and cardiolipin due to lipid peroxidation, BIOCHEM.BIOPHYS. RES. COMM. 264:343-347 (1999). This leads to the release of thecytochrome Cinto the mitochondrial intermembrane space, inducingapoptotic cell death.

Further, the peroxidation of CL has the effect of opening themitochondrial permeability transition pore (“MPTP”), see Dolder, M. etal., Mitochondria creatine kinase in contact sites: Interaction withporin and adenine nucleotide translocase, role in permeabilitytransition and sensitivity to oxidative damage, BIOL. SIGNALS RECEPT.,10:93-111 (2001); also Imai, H. et al., Protection from inactivation ofthe adenine nucleotide translocator during hypoglycaemia-inducedapoptosis by mitochondria/phospholipid hydroperoxide glutathioneperoxidase, BIOCHEM. J., 371:799-809 (2003). Accordingly, themitochondrial membrane swells and releases the cytochrome C into thecytosol. Excess cytochrome C in the cytosol leads to cellular apoptosis,see Iverson, S. L. et al. The cardiolipin-cytochrome c interaction andthe mitochondria regulation of apoptosis, ARCH. BIOCHEM. BIOPHYS.423:37-46 (2003).

Moreover, mitochondrial dysfunction and cell death may ultimately leadto multiple organ failure despite resuscitative efforts or supplementaloxygen supply, see Cairns, C., Rude Unhinging of the Machinery of Life:Metabolic approaches to hemorrhagic Shock, CURRENT CRITICAL CARE, 7:437(2001). Accordingly, there is a need in the art for an antioxidant mimicsimilar to SOD which scavenges the ROS, thereby reducing oxidativestress. Reduction of oxidative stress delays, even inhibits,physiological conditions that otherwise might occur, such as hypoxia.

Also, there is also a need to improve the permeability of antioxidants'penetration of the cellular membrane. One of the limitations of SOD isthat it cannot easily penetrate the cell membrane. However, nitroxideradicals, such as TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) and itsderivatives, have been shown to penetrate the cell membrane better thanSOD. Further, nitroxide radicals like TEMPO prevent the formation ofROS, particularly superoxide, due to their reduction by themitochondrial electron transport chain to hydroxyl amine radicalscavengers, see Wipf P. et al., Mitochondria targeting of selectiveelectron scavengers: synthesis and biological analysis ofhemigramicidin-TEMPO conjugates, J. AM. CHEM. SOC. 127:12460-12461.Accordingly, selective delivery of TEMPO derivatives may lead to atherapeutically beneficial reduction of ROS and may delay or inhibitcell death due to the reduction of oxidative stress on the cell.

This selective delivery may be accomplished by way of a number ofdifferent pathways—i.e., a biological or chemical moiety has a specifictargeting sequence for penetration of the cell membrane, ultimatelybeing taken up by the mitochondrial membrane. Selective delivery of anitroxide SOD mimic into the mitochondrial membrane has provendifficult. Accordingly, there is a need in the art for effective andselective delivery of TEMPO antioxidant derivatives that specificallytarget the mitochondrial membrane to help reduce the ROS and RNSspecies. Said antioxidants also help prevent cellular and mitochondriaapoptotic activity which often results due to increased ROS species, seeKelso et al., Selective Targeting of a Redox-active Ubiquinone toMitochondria within Cells: Antioxidant and Antiapoptotic Properties, J.BIOL. CHEM., 276: 4588 (2001).

U.S. Patent Application 2005/0169904 discloses a conjugate whichcomprises the following: (i) a mitochondrial membrane-permeant peptide;(ii) a mitochondrial-active agent or compound of interest such as adetectable group or compound, an active mitochondrial protein orpeptide, nucleic acids, drug or signaling agent; and, (iii) amitochondrial targeting sequence linking said mitochondrialmembrane-permeant peptide and said active mitochondrial protein orpeptide. The targeting sequence of the conjugate is cleaved within themitochondrial matrix, not within the cellular cytoplasm of a target cellinto which said mitochondrial-active agent or compound is to bedelivered. Methods of use of these compounds and agents are alsodisclosed within the publication. A disadvantage of this metholodologyis that it requires cleavage of the peptide sequence in order to releasethe active agent.

U.S. Pat. No. 6,331,532 and U.S. Patent Application 2005/0245487 A1disclose mitochondrially targeted antioxidant compounds. The compoundcomprises a lipophilic cation covalently bonded to an antioxidantmoiety. Pharmaceutical compositions containing the mitochondriallytargeted antioxidant compounds, and methods of therapy or prophylaxis ofpatients who would benefit from reduced oxidative stress are disclosed.This methodology relies on ionic or lipophilic interactions and is lessselective than the present invention.

U.S. Patent Application 2005/0107366 A1 discloses a pharmaceuticalcomposition that is covalently bound to a non-toxic spin trappingcompound. Spin trapping compositions generally have been known to beeffective in treating a variety of disorders. Spin trapping compoundsare molecules that have an unpaired electron (i.e., paramagnetic), forma stable compound or complex with a free radical, and lack cytotoxicity.One example of a spin trapping compound is TEMPO. These spin trappingcompounds, such as TEMPO, provide a unique signal that can be measuredby electron spin spectroscopy (“ESR”). Since an effectivemitochondrial-targeting sequence is not used, this approach is not asefficient as the present invention.

TEMPO and its derivatives are antioxidants that have been shown toimprove physiologic variables after induced hemorrhagic shock, such asheart rate, systolic blood pressure, acid-base balance, serumantioxidant status, and survival time, see Kentner et al., EarlyAntioxidant Therapy with TEMPOL during Hemorrhagic Shock IncreasesSurvival in Rats, J. OF TRAUMA® INJURY, INFECTION, AND CRITICAL CARE,968 (2002). In general, effective levels of administered TEMPO are toohigh to accomplish therapeutic effects.

Therefore, in spite of the foregoing prior art, there remains a veryreal need for a composition and associated methods for delivering cargoof various types to mitochondria. In one embodiment, a compositioncomprising membrane active peptidyl fragments having a high affinitywith the mitochondria linked to cargo is provided. The cargo may beselected from a large group of candidates. The invention alsocontemplates compositions and methods for effectively treating acondition that is caused by excessive mitochondria production of ROS andRNS in the mitochondrial membrane.

SUMMARY OF THE INVENTION

The present invention has met the hereinbefore described need byproviding compositions and methods as disclosed for treating acondition, including a disease or other medical condition, which iscaused by excessive mitochondrial production of ROS in the mitochondrialmembrane.

In its broader aspects, the invention contemplates a composition fordelivering cargo to a mitochondria comprising (a) a membrane activepeptidyl fragment having a high affinity with the mitochondria and (b) acargo. The composition may have a property selected from the groupconsisting of antioxidant, radioprotective, protective, anti-apoptotic,therapeutic, ameliorative, NOS antagonist and combinations thereof. Thecomposition may be selected from the group consisting of XJB-5-234,XJB-5-133, XJB-5-241, and XJB-5-127. Other compositions as hereindescribed may be employed.

The membrane active peptidyl fragment may have a β-turn motif.

In one embodiment, the membrane active peptidyl fragment may be from anantibiotic molecule that acts by targeting the bacterial cell wall. Awide variety of cargos may be selected for use in the composition andmethod of the invention. One category of cargo employable in theinvention is a radical scavenging agent. Another category of cargo is anNOS antagonist.

In a further embodiment of the invention, a patient is treated byadministering to the patient a composition, and effecting by saidadministration inhibiting patient cells from injury. The composition has(a) a membrane active peptidyl fragment having a high affinity withmitochondria and (b) cargo.

In a preferred embodiment, the compositions are conjugates of GramicidinS peptidyl fragments and TEMPO derivatives. The Gramicidin S peptidylfragment is either Gramicidin S or a hemigramicidin fragment. In afurther embodiment, a hemigramicidin (i.e., a fragmented version ofGramicidin S with additional functional groups) motif with an amide bondisostere constitute the Gramicidin moiety of the TEMPO-peptidylconjugate. In a preferred embodiment, the amide bond isostere is an(E)-alkene moiety.

In yet another embodiment, the mitochondrial targeting agent is a GSpeptidyl fragment which comprises peptide bond mimetics.

Yet another embodiment provides a method for delivering antioxidants,such as TEMPO, for example, into cells to mitochondria. Specifically,the peptidyl conjugates comprise a targeting sequence which isrecognizable by the mitochondria and also permeable to the mitochondriamembrane. The peptidyl conjugate thereby “anchors” the antioxidant“payload” into the mitochondrial membrane whereby the antioxidant actsas an electron scavenger of the ROS present within the membrane.Accordingly, the electron scavenging activity of the antioxidant helpsresist mitochondrial dysfunction and cell death.

Another preferred embodiment provides a method for therapeuticallyadministering the TEMPO-peptidyl conjugate to patients with hemorrhagicshock to help prolong survival until it is feasible to obtain control ofthe bleeding vessels of the patient. Another related embodiment providesa method for therapeutically administering the TEMPO-peptidyl conjugateto patients with hemorrhagic shock to help prolong survival until it isfeasible to obtain control of the bleeding vessels of the patient, inspite of a lack of resuscitation with blood or other non-sanguineousfluids. Yet another related embodiment provides a method fortherapeutically administering the TEMPO-peptidyl conjugate to patientswith hemorrhagic shock to help prolong survival until it is feasible toobtain control of the bleeding vessels of the patient, in spite ofhypotension.

The invention also contemplates a composition for scavenging theradicals in a mitochondrial membrane comprising a radical scavengingagent and a membrane active compound having a high affinity with themitochondria. It also contemplates a related method for delivering acomposition to mitochondria comprising transporting to saidmitochondrial membrane (a) a radical scavenging agent and (b) a membraneactive compound having a high affinity with the mitochondrial membrane.

In another embodiment, the cargo which is covalently linked to themitochondrial-selective targeting agents may be an inhibitor of nitricoxide synthase (“NOS”) enzyme activity.

An object of this invention is to provide compositions which effectivelyact as mitochondria-selective targeting agents to help deliver anattached moiety to the mitochondria membrane.

Another object of this invention is to provide compositions transportedby mitochondrial-selective targeting agents which may include aninhibitor of NOS enzyme activity

Another object of this invention is to provide a method for deliveringthese agents effectively into cells and mitochondria where they act aselectron scavengers.

Yet another object of the invention is to provide a method whichprovides agents that provide protection against mitochondrialdysfunction and cell apoptosis.

Yet another object of this invention is to prolong the survival of apatient that has suffered hemorrhagic shock.

A related object of this invention is to prolong the survival of apatient that has suffered hemorrhagic shock and has not beenresuscitated with blood or non-sanguineous fluids.

Yet another related object of this invention is to prolong the survivalof a patient that has suffered hemorrhagic shock and is hypotensive.

Still another object of the invention is to provide a composition andmethod for treating a condition, including a disease or other medicalcondition, which is the result of excessive mitochondrial production ofROS.

Yet another object of this invention is to provide a method foradministering the composition to patients with a condition, including adisease or an illness, which is the result from excessive mitochondrialproduction of ROS.

These and other objects of the invention will be more fully understoodfrom the following detailed description of the invention on reference tothe illustrations and table appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structure of TEMPOL and the sevenhemigramicidin derivative compounds. Panel A shows TEMPOL. Panel B showsa dipeptidic TEMPO analog—XJB-5-208. Panel C shows ahemigramicidin-TEMPO conjugate—XJB-5-125. Panel D shows a hemigramicidinconjugate that has AMT functional moiety in place of the TEMPOmoiety—XJB-5-127. Panel E shows a hemigramicidin-TEMPOconjugate—XJB-5-131. Panel F shows a hemigramicidin conjugate that hasthe AMT functional moiety in place of the TEMPO moiety—XJB-5-133. PanelG shows a hemigramicidin-TEMPO conjugate—XJB-5-197. Panel H shows ahemigramicidin compound that does not have the TEMPO or AMTmoieties—XJB-5-194.

FIG. 2 depicts an example of a synthetic pathway for theTEMPO-hemigramicidin conjugates.

FIG. 3 shows (A) an EPR-based analysis of integration and (B) reductionof nitroxide Gramicidin S peptidyl-TEMPO conjugates in MECs.

FIG. 4A-4D shows a flourescein isothiocyanate-dextran (FD4) read-outwhich reflects the effect of Gramicidin-S TEMPO conjugates on rat ilealmucosal permeability following profound hemorrhagic shock. Data areexpressed as a percentage of the change permeability relative to thatobserved in simultaneously assayed control segments loaded during shockwith normal saline solution. FIG. 4A, panel A shows an FD4 read-out ofTEMPOL which is used as a “positive control” for the gut mucosalprotection assay. FIG. 4A, panel B shows an FD4 read-out of TEMPOconjugate XJB-5-208 reflecting gut mucosal protection. FIG. 4B, panel Cshows an FD4 read-out of XJB-5-125 which has the TEMPO payload, butfails to provide protection against gut barrier dysfunction induced byhemorrhage. FIG. 4B, panel D shows an FD4 read-out of XJB-5-127 whichlacks the TEMPO payload and fails to provide protection against gutbarrier dysfunction induced by hemorrhage. FIG. 4C, panel E shows an FD4read-out of TEMPO conjugate XJB-5-131 reflecting gut mucosal protection.FIG. 4C, panel F shows an FD4 read-out of XJB-5-133 which lacks theTEMPO payload even though it possesses the same hemigramicidinmitochondria targeting moiety as the most active compound, XJB-5-131.FIG. 4D, panel G shows an FD4 read-out of XJB-5-197 which has the TEMPOpayload, but fails to provide protection against gut barrier dysfunctioninduced by hemorrhage. FIG. 4D, panel H shows an FD4 read-out ofXJB-5-194 which lacks the TEMPO payload and fails to provide protectionagainst gut barrier dysfunction induced by hemorrhage.

FIG. 5 shows graphical representations of the effect of nitroxideconjugates on ActD-induced apoptosis. Panel A is a graphicalrepresentation of superoxide production based upon mean fluorescenceintensity from 10,000 ileal cells. Panel B is a graphical representationof phosphatidylserine (PS) externalization as indicated by thepercentage of annexin V-positive cells. Panel C is a graphicalrepresentation of caspase-3 activity as indicated by amount of itsspecific substrate present, Z-DVED-AMC, in nmol/mg protein. Panel D is agraphical representation of DNA fragmentation as indicated by propidiumiodide fluorescence. Panel E is a graphical representation of PSexternalization at different concentrations of the compound 5a. Panel Fis a graphical representation of adenosine triphosphate (ATP) levels inmitochondria in the presence or absence of 5a or 2-deoxyglucose.

FIG. 6 illustrates the effects of intraluminal XJB-5-131 onhemorrhage-induced peroxidation of phospholipids in intestinal mucosa.The upper left panel is a graphical representation of the peroxidationof phosphatidylcholine (“PC”). The upper right panel is a graphicalrepresentation of peroxidation activity with respect tophosphatidylethanolamine (“PE”). The lower left panel is a graphicalrepresentation of peroxidation activity with respect tophosphatidylserine (“PS”). The lower right panel is a graphicalrepresentation of peroxidation activity with respect to cardiolipin(“CL”).

FIG. 7 is a graphical representation of caspase 3 and 7 activity thatillustrates the effects of intraluminal XJB-5-131.

FIG. 8 is a graphical representation of permeability of XJB-5-131 withrespect to Caco-2_(BBe) human enterocyte-like monolayers subjected tooxidative stress. The permeability of the monolayers is expressed as aclearance (pL·h⁻¹·cm²).

FIG. 9A-9B is a graphical representation of the effects of intravenoustreatment with XJB-5-131 on MAP (mean arterial pressure, mm Hg) of ratessubjected to volume controlled hemorrhagic shock. FIG. 9B is a graphicalrepresentation of the effects of intravenous treatment with XJB-5-131 onsurvival probability of rates subjected to volume controlled hemorrhagicshock.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “patient” refers to members of the animalkingdom including but not limited to human beings.

As used herein, the term “reactive oxygen species” (“ROS”) includes, butis not limited to, superoxide anion, hydroxyl, and hydroperoxideradicals.

The present invention relates to compositions and methods for providingmitochondria-selective targeting agents bonded, as by covalent linking,for example, to desired cargo such as radical scavenging agents, forexample.

In another embodiment, the cargo transported by mitochondria-selectivetargeting agents may include an inhibitor of NOS activity.

While the generation of ROS in small amounts is a typical byproduct ofthe cellular respiration pathway, certain conditions, including adisease or other medical condition, may occur in the patient when theamount of ROS is excessive to the point where natural enzyme mechanismscannot scavenge the amount of ROS being produced.

The present invention provides compositions and methods that scavengereactive oxygen species that are present within the mitochondrialmembrane of the cell. These compositions and methods have the utility ofbeing able to scavenge an excess amount of ROS being produced thatnaturally occurring enzymes SOD and catalase, among others, cannot copewith.

In a preferred embodiment of the invention, a composition for deliveringcargo to a mitochondria comprises (a) a membrane active peptidylfragment having a high affinity with mitochondria and (b) cargo. Arelated method which employs the delivery to mitochondria by means of amembrane active peptidyl fragment having a high affinity with themitochondria is also contemplated.

The cargo may have a property selected from the group consisting ofantioxidant, radioprotective, protective, anti-apoptotic, therapeutic,ameliorative, NOS antagonist and combinations thereof. The cargo may beselected from the group consisting of XJB-5-234, XJB-5-133, XJB-5-241,and XJB-5-127. It is preferred that the membrane active peptidyl have aβ-turn motif and that the active peptidyl fragment is bonded to thecargo.

In one embodiment, the membrane active peptidyl fragment may be derivedfrom an antibiotic. The fragment may be from an antibiotic molecule thatacts by targeting the bacterial cell wall. The antibiotic is preferablyselected from the group consisting of bacitracins, gramicidins,valinomycins, enniatins, alamethicins, beauvericin, serratomolide,sporidesmolide, tyrocidins, polymyxins, monamycins, and lissoclinumpeptides.

The cargo may be categorized by the property of creating biologicaleffects which endow the mitochondria with resistance to oxidative damageinduced by free radicals. The cargo may be a radical scavenging agent.In one embodiment, the cargo may be selected from the group consistingof TEMPO and TEMPO moieties.

In another embodiment, the cargo may be categorized by the property ofendowing the mitochondria with resistance to nitrosative damage.

In another embodiment, the cargo may be categorized by the property ofinhibiting nitric oxide synthase enzyme activity. It will be appreciatedthat a wide variety of cargoes may be employed in the composition andwith the method of the invention. Among the additionally preferredcargos are a cargo selected from the group consisting of a2-amino-6-methyl-thiazine, a ubiquinone analog, a ubiquinone analogfragment moiety, a ubiquinone analog fragment moiety lacking ahydrophilic tail, a superoxide dismutase mimetic, a superoxide dismutasebiomimetic and a salen-manganese compound.

In a therapeutic embodiment of the invention, a patient may be treatedby administering to the patient a composition which inhibits patientcells injury. The cargo employed in this therapeutic method may serve toscavenge free radicals. In another embodiment of the method, the cargomay serve to inhibit nitric oxide synthase.

In one aspect of the invention, compounds possessing electron scavengerproperties similar to SOD and catalase are disclosed. In another aspectof the invention, methods for targeting the mitochondria of the cell arediscussed.

In one embodiment of the present invention, the compositions werederived from a covalent form of 4-amino-TEMPO and peptidyl fragments ofGramicidin S. As is known to one ordinarily skilled in the art,Gramicidin S is a well-known antibiotic and has a high affinity tobacterial membranes. Further, bacteria and mitochondria are structurallyquite similar. Accordingly, it was found that appropriately foldedpeptidyl fragments of Gramicidin S, such as those having a β-turn, havea high affinity for mitochondria and are introduced into themitochondria membrane and permeate through the cytoplasm membrane of thecell. Therefore, targeting of mitochondria by way of Gramicidin Speptidyl fragments proved to be useful. In another embodiment, thecompositions were derived from a conjugate form of TEMPOL and peptidylfragments of Gramicidin S.

Accordingly, using the Gramicidin S peptidyl fragments and alkeneisosteres (beta turns) as “anchors,” the TEMPO “payload” could be guidedinto the mitochondrial membrane.

In one embodiment, the Leu-^(D)Phe-Pro-Val-Orn fragment ofhemigramicidin may advantageously be used as a targeting sequence. Theβ-turn motif of the Gramicidin-S fragment directs most of the polarfunctionality of the peptide strand into the core; the amino functionalgroups of Leu and Orm are acylated so that cytotoxicity of Gramicidin Sis reduced.

Alkene isosteres such as (E)-alkene isosteres of Gramicidin S (i.e.,hemigramicidin) were used as part of the targeting sequence. See FIG. 2for a synthetic pathway for (E)-alkene isosteres and reference number 2for the corresponding chemical structure. First, hydrozirconation ofalkyne (FIG. 2, compound 1) with Cp₂ZrHCl is followed by transmetalationto Me₂Zn and the addition of N-Boc-isovaleraldimine. The resultingcompound (not shown) was then worked up using a solution oftetrabutylammonium fluoride (“TBAF”) and diethyl ether with a 74% yield.The resulting compound was then treated with acetic anhydride,triethylamine (TEA), and 4-N,N¹-(dimethylamino) pyridine (“DMAP”) toprovide a mixture of diastereomeric allylic amides with a 94% yieldwhich was separated by chromatography. Finally, the product was workedup with K₂CO₃ in methanol to yield the (E)-alkene, depicted as compound2.

The (E)-alkene, depicted as compound 2 of FIG. 2, was then oxidized in amulti-step process to yield the compound 3 (FIG. 2)—an example of the(E)-alkene isostere.

Then, the compound 3 of FIG. 2 was then conjugated with the peptideH-Pro-Val-Orn (Cbz)-OMe using 1-ethyl-3-(3-dimethylaminopropylcarbodimide hydrochloride) (EDC) as a coupling agent. The peptide is anexample of a suitable targeting sequence having affinity for themitochondria of a cell. The resulting product is shown as compound 4a inFIG. 2. Saponification of compound 4a followed by coupling with4-amino-TEMPO (4-AT) afforded the resulting conjugate shown as compound5a in FIG. 2, in which the Leu-^(D)Phe peptide bond has been replacedwith an (E)-alkene.

In an alternate embodiment, conjugates 5b in FIG. 2 was prepared bysaponification and coupling of the peptide 4b(Boc-Leu-^(D)Phe-Pro-Val-Orn(Cbz)-OMe) with 4-AT. Similarly, conjugate5c in FIG. 2 was prepared by coupling the (E)-alkene isostere asindicated as compound 3 in FIG. 2 with 4-AT. These peptide and peptideanalogs are additional examples of suitable targeting sequences havingan affinity to the mitochondria of a cell.

In another embodiment of the invention, peptide isosteres may beemployed as the conjugate. Among the suitable peptide isosteres aretrisubstituted (E)-alkene peptide isosteres and cyclopropane peptideisosteres, as well as all imine addition products of hydro- orcarbometalated internal and terminal alkynes for the synthesis of di andtrisubstituted (E)-alkene and cyclopropane peptide isosteres. See Wipfet al. Imine additions of internal alkynes for the synthesis oftrisubstituted (E)-alkene and cyclopropane isosteres, ADV. SYNTH. CATAL.347, 1605-1613 (2005). These peptide mimetics have been found to act asβ-turn promoters. See Wipf et al. Convergent Approach to (E)-Alkene andCyclopropane Peptide Isosteres, Organic Letters, VOL. 7, No. 103-106(2005).

As is known to one ordinarily skilled in the art, nitroxide andnitroxide derivatives, including TEMPOL and associated TEMPO derivativesare stable radicals that can withstand biological environments.Therefore, the presence of the 4-amino-TEMPO or the TEMPOL “payload”within the mitochondria membrane can serve as an effective and efficientelectron scavenger of the ROS being produced within the membrane.

Materials and Methods Examples Materials

All chemicals were from SIGMA-ALDRICH (St Louis, Mo.) unless otherwisenoted. Heparin, ketamine HCl and sodium pentobarbital were from ABBOTTLABORATORIES (North Chicago, Ill.). Dulbecco's modified Eagle medium(“DMEM”) was from BIOWHITTAKER (Walkersville, Md.). Fetal bovine serum(FBS; <0.05 endotoxin units/ml) was from HYCLONE (Logan, Utah).Pyrogen-free sterile normal saline solution was from BAXTER (Deerfield,Ill.).

General.

All moisture-sensitive reactions were performed using syringe-septum captechniques under an N2 atmosphere and all glassware was dried in an ovenat 150° C. for 2 h prior to use. Reactions carried out at −78° C.employed a CO₂-acetone bath. Tetrahydrofuran (THF) was distilled oversodium/benzophenone ketyl; CH₂Cl₂, toluene and Et₃N were distilled fromCaH₂. Me₂Zn was purchased from ALDRICH COMPANY.

Reactions were monitored by thin layer chromatography (“TLC”) analysis(EM SCIENCE pre-coated silica gel 60 F254 plates, 250 μm layerthickness) and visualization was accomplished with a 254 nm UV light andby staining with a Vaughn's reagent (4.8 g (NH₄)₆Mo7O₂₄.4H₂O, 0.2 gCe(SO₄)₂.4H₂O in 10 mL conc. H₂SO₄ and 90 mL H₂O). Flash chromatographyon SiO₂ was used to purify the crude reaction mixtures.

Melting points were determined using a LABORATORY DEVICES Mel-Temp II.Infrared spectra were determined on a NICOLET Avatar 360 FT-IRspectrometer. Mass spectra were obtained on a WATERS Autospec doublefocusing mass spectrometer (“EI”) or a WATERS Q-Tof mass spectrometer(“ESI”). LC-MS data were obtained on an Agilent 1100 instrument, using aWATERS Xterra MS CH 3.5 μm RP column (4.6×100 mm).

Synthesis, Example I

Prepared as a colorless oil (FIG. 2, compound 1) according to theliterature procedure, see Edmonds, M. K. et al. Design and Synthesis ofa Conformationally Restricted Trans Peptide Isostere Based on theBioactive Conformations of Saquinavir and Nelfinavir J. ORG. CHEM.66:3747 (2001); see also Wipf P. et al., Convergent Approach to(E)-Alkene and Cyclopropane Peptide Isosteres ORG. LETT. 7:103 (2005);see also Xiao, J. et al., Electrostatic versus Steric Effects inPeptidomimicry: Synthesis and Secondary Structure Analysis of GramicidinS Analogues with (E)-Alkene Peptide Isosteres J. AM. CHEM. SOC. 127:5742(2005).

A solution of 2.20 g (5.52 mmol) of compound 1 (FIG. 2) in 20.0 mL ofdry CH₂Cl₂ was treated at room temperature with 1.85 g (7.17 mmol) ofCp₂ZrHCl. The reaction mixture was stirred at room temperature for 5min, CH₂Cl₂ was removed in vacuo and 20.0 mL of toluene was added. Theresulting yellow solution was cooled to −78° C. and treated over aperiod of 30 min with 2.76 mL (5.52 mmol) of Me₂Zn (2.0 M solution intoluene). The solution was stirred at −78° C. for 30 min, warmed to 0°C. over a period of 5 min and treated in one portion with 2.05 g (11.1mmol) of N-Boc-isovaleraldimine, see Edmonds, M. K. et al. Design andSynthesis of a Conformationally Restricted Trans Peptide Isostere Basedon the Bioactive Conformations of Saquinavir and Nelfinavir J. ORG.CHEM. 66:3747 (2001); see also Wipf P. et al., Convergent Approach to(E)-Alkene and Cyclopropane Peptide Isosteres J. ORG. LETT. 7:103(2005); see also Xiao et al., Electrostatic versus Steric Effects inPeptidomimicry: Synthesis and Secondary Structure Analysis of GramicidinS Analogues with (E)-Alkene Peptide Isosteres J. AM. CHEM. SOC. 127:5742(2005).

The resulting mixture was stirred at 0° C. for 2 h, quenched withsaturated NH₄Cl, diluted with EtOAc, filtered through a thin pad ofCelite, and extracted with EtOAc. The organic layer was dried (MgSO₄),concentrated in vacuo, and purified by chromatography on SiO₂ (20:1,hexane/EtOAc) to yield 3.13 g (97%) as a colorless, oily 1:1 mixture ofdiastereomers.

A solution of 4.19 g (7.15 mmol) of product in 100 mL of drytetrahydrofuran (“THF”) was treated at 0° C. with 9.30 mL (9.30 mmol) oftetrabutylammoniumflouride (TBAF, 1.0 M solution in THF). The reactionmixture was stirred at room temperature for 20 h, diluted with EtOAc,and washed with brine. The organic layer was dried (MgSO₄), concentratedin vacuo, and purified by chromatography on SiO₂ (4:1, hexane/EtOAc) toyield 1.89 g (76%) as a light yellowish, foamy 1:1 mixture ofdiastereomers.

A solution of 1.86 g (5.23 mmol) of product in 40.0 mL of dry CH₂Cl₂ wastreated at 0° C. with 1.46 mL (10.5 mmol) of triethylamine (“TEA”), 2.02mL (21.4 mmol) of Ac₂O, and 63.9 mg (0.523 mmol) of4-N,N¹-(dimethylamino) pyridine (“DMAP”). The reaction mixture wasstirred at 0° C. for 15 min and at room temperature for 3 h, dilutedwith EtOAc, and washed with brine. The organic layer was dried (MgSO₄),concentrated in vacuo, and purified by chromatography on SiO₂ (20:1,hexane/Et₂O) to yield 1.97 g (94%) of acetic acid(2S)-benzyl-(5R)-tert-butoxycarbonylamino-7-methyloct-(3E)-enyl ester(807 mg, 38.7%), acetic acid(2S)-benzyl-(5S)-tert-butoxycarbonylamino-7-methyloct-(3E)-enyl ester(826 mg, 39.6%), and a mixture of the aforementioned species (337 mg,16.2%).

A solution of 350 mg (0.899 mmol) of acetic acid(2S)-benzyl-(5S)-tert-butoxycarbonylamino-7-methyloct-(3E)-enyl ester in8.00 mL of MeOH was treated at 0° C. with 62.0 mg (0.449 mmol) of K₂CO₃.The reaction mixture was stirred at 0° C. for 1 h and at roomtemperature for an additional 4 h, diluted with EtOAc, and ashed withH₂O. The organic layer was dried (MgSO₄), concentrated in vacuo, andpurified by chromatography on SiO₂ (4:1, hexane/EtOAc) to yield 312 mg(quant.) of compound 2 (FIG. 2) as a colorless oil.

A solution of 23.0 mg (66.2 μmol) of compound 2 (FIG. 2) in 2.00 mL ofdry CH₂Cl₂ was treated at 0° C. with 42.1 mg (99.3 μmol) of Dess-MartinPeriodinane. The reaction mixture was stirred at 0° C. for 1 h and atroom temperature for an additional 4 h, quenched with saturated Na₂S₂O₃in a saturated NaHCO₃ solution, stirred for 30 min at room temperature,and extracted with CH₂Cl₂. The organic layer was dried (Na₂SO₄),concentrated in vacuo to give a colorless foam and subsequentlydissolved in 3.00 mL of THF, and treated at 0° C. with 300 μL (600 μmol)of 2-methyl-2-butene (2.0 M solution in THF) followed by anothersolution of 18.0 mg (199 μmol) of NaClO₂ and 18.2 mg (132 μmol) ofNaH₂PO₄.H₂O in 3.00 mL of H₂O. The reaction mixture was stirred at 0° C.for 1 h and at room temperature for an additional 3 h, extracted withEtOAc, and washed with H₂O. The organic layer was dried (Na₂SO₄) andconcentrated in vacuo to yield compound 3 (FIG. 2) as a crude colorlessfoam that was used for the next step without purification.

A solution of crude compound 3 (FIG. 2) (66.2 μmol) in 3.00 mL of CHCl₃was treated at 0° C. with 10.7 mg (79.2 μmol) of 1-hydroxybenzotrizole(“HOBt”) and 14.0 mg (73.0 μmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride (“EDC”), followed by a solution of 62.9 mg(132 μmol) of H-Pro-Val-Orn(Cbz)-OMc, see Edmonds, M. K. et al. Designand Synthesis of a Conformationally Restricted Trans Peptide IsostereBased on the Bioactive Conformations of Saquinavir and Nelfinavir J.ORG. CHEM. 66:3747 (2001); see also Wipf P. et al., Convergent Approachto (E)-Alkene and Cyclopropane Peptide Isosteres J. ORG. LETT. 7:103(2005); see also Xiao, J. et al., Electrostatic versus Steric Effects inPeptidomimicry: Synthesis and Secondary Structure Analysis of GramicidinS Analogues with (E)-Alkene Peptide Isosteres J. AM. CHEM. SOC. 127:5742(2005), in 1.00 mL of CHCl₃ and 0.8 mg (6.6 μmol) of DMAP. The reactionmixture was stirred at room temperature for 2 d, diluted with CHCl₃, andwashed with H₂O. The organic layer was dried (Na₂SO₄), concentrated invacuo, and purified by chromatography on SiO₂ (from 2:1, hexanes/EtOActo 20:1, CHCl₃/MeOH) to yield 51.3 mg (94%) of compound 4a (FIG. 2) as acolorless foam.

A solution of 53.7 mg (65.5 μmol) of compound 4a (FIG. 2) in 2.00 mL ofMeOH was treated at 0° C. with 655 μL (655 μmol) of 1 N NaOH. Thereaction mixture was stirred at room temperature for 6 h, and treatedwith 655 μL (655 μmol) of 1 N HCl. The solution was extracted with CHCl₃and the organic layer was dried (Na₂SO₄) and concentrated in vacuo togive the crude acid as a colorless form. This acid was dissolved in 5.00mL of CHCl₃ and treated at room temperature with 10.6 mg (78.4 μmol) ofHOBt, 15.1 mg (78.8 μmol) of EDC, 20.2 mg (118 μmol) of 4-amino-TEMPOand 8.0 mg (65.5 μmol) of DMAP. The reaction mixture was stirred at roomtemperature for 36 h, diluted with CHCl₃, and washed with H₂O. Theorganic layer was dried (Na₂SO₄), concentrated in vacuo, and purified bychromatography on SiO₂ (from 1:1, hexane/EtOAc to 20:1, CHCl₃/MeOH) toyield 62.0 mg (99%) of compound 5a (FIG. 2) as a colorless solid. Thefollowing characterization data were obtained: LC-MS (Rt 8.81 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=959.5[M+H]⁺, 981.5 [M+Na]⁺) and HRMS (ESI) m/z calculated for C₅₃H₈₀N₇O₉Na(M+Na) 981.5915. found 981.5956.

A solution of 60.0 mg (71.7 μmol) of compound 4b (FIG. 2), see Tamaki,M. et al. I. BULL. CHEM. SOC. JPN, 66:3113 (1993), in 2.15 mL of MeOHwas treated at room temperature with 717 tL (717 μmol) of 1 N NaOH. Thereaction mixture was stirred at room temperature for 5 h, and treated at0° C. with 717 μL (717 μmol) of 1 N HCl. The solution was extracted withCHCl₃ and the organic layer was dried (Na₂SO₄) and concentrated in vacuoto give the crude acid as colorless foam. The acid was dissolved in 6.04mL of CHCl₃ and treated at room temperature with 11.6 mg (85.8 μmol) ofHOBt, 16.5 mg (85.1 μmol) of EDC, 18.5 mg (108 μmol) of 4-amino-TEMPOand 8.8 mg (72.0 μmol) of DMAP. The reaction mixture was stirred at roomtemperature for 20 h, diluted with CHCl₃, and washed with H₂O. Theorganic layer was dried (Na₂SO₄), concentrated in vacuo, and purified bychromatography on SiO₂ (from 2:1, hexane/EtOAc; to 20:1, CHCl₃/MeOH) toyield 69.6 mg (99%) of compound 5b (FIG. 2) as a yellowish solid. Thefollowing characterization data were obtained: LC-MS (Rt 7.02 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=976.5[M+H]′, 998.4 [M+Na]⁺) and HRMS (ESI) m/z calculated for C₅₂H₇₉N₈O₁₀Na(M+Na) 998.5817. found 998.5774.

A solution of crude compound 3 (FIG. 2) (40.3 μmol) in 3.00 mL of CH₂Cl₂was treated at 0° C. with 10.4 mg (60.7 μmol) of 4-amino-TEMPO, 7.7 mg(40.2 μmol) of EDC, and 5.4 mg (44.2 μmol) of DMAP. The reaction mixturewas stirred at room temperature for 20 h, diluted with CHCl₃, and washedwith H₂O. The organic layer was dried (Na₂SO₄), concentrated in vacuo,and purified by chromatography on SiO₂ (from 4:1 to 1:1, hexane/EtOAc)to yield 18.8 mg (91%) of compound 5c (FIG. 2) as a yellowish solid. Thefollowing characterization data were obtained: LC-MS (Rt 7.01 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=537.3[M+Na]⁺) and HRMS (ESI) in/z calculated for C30H48N3O4Na (M+Na)537.3543. found 537.3509.

Determination of Intracellular Superoxide Radicals

Oxidation-dependent fluorogenic dye, dihydroethidium (“DHE”, MolecularProbes) was used to evaluate intracellular production of superoxideradicals. DHE is cell permeable and, in the presence of superoxide, isoxidized to fluorescent ethidium which intercalates into DNA. Thefluorescence of ethidium was measured using a FACscan (BECTON-DICKINSON,Rutherford, N.J.) flow cytometer, equipped with a 488-nm argon ion laserand supplied with the Cell Quest software. Mean fluorescence intensityfrom 10,000 cells were acquired using a 585-nm bandpass filter (FL-2channel).

Determination of Intracellular ATP Levels.

Cells were incubated with 10 μm of compound 5a (FIG. 2) for indicatedperiods of time (2, 4, 6, 12, and 14 h). At the end of incubation, cellswere collected and the content of intracellular ATP was determined usinga bioluminescent somatic cell assay kit (SIGMA, St. Louis, Mass.). As apositive control, cells were incubated with 2 mM of 2-dexy-glucose, aglucose analogue which competitively inhibits cellular uptake andutilization of glucose, for 12 and 14 h.

Cells.

Caco-2BBe human enterocyte-like epithelial cells were obtained from theAMERICAN TYPE CULTURE COLLECTION (Manassas, Va.). Cells were routinelymaintained at 37° C. in under a humidified atmosphere containing 8% CO2in air. The culture medium was DMEM supplemented with 10% FBS,non-essential amino acids supplement (SIGMA-ALDRICH catalogue #M7145),sodium pyruvate (2 mM), streptomycin (0.1 mg/ml), penicillin G (100U/ml) and human transferrin (0.01 mg/ml). The culture medium was changed3 times per week.

Surgical Procedures to Obtain Vascular Access.

All study protocols using rats followed the guidelines for the use ofexperimental animals of the US National Institutes of Health and wereapproved by the Institutional Animal Care and Use Committee at theUniversity of Pittsburgh.

Male specific pathogen-free Sprague Dawley rats (CHARLES RIVERLABORATORIES, Wilmington, Mass.), weighing 150-250 g, were housed in atemperature-controlled environment with a 12-h light/dark cycle. Therats had free access to food and water. For experiments, rats wereanesthetized with intramuscular ketamine HCl (30 mg/kg) andintraperitoneal sodium pentobarbital (35 mg/kg). Animals were kept in asupine position during the experiments. Lidocaine (0.5 ml of a 0.5%solution) was injected subcutaneously to provide local anesthesia atsurgical cut-down sites. In order to secure the airway, a tracheotomywas performed and polyethylene tubing (PE 240; BECTON-DICKINSON, Sparks,Md.) was introduced into the trachea. Animals were allowed to breathespontaneously.

The right femoral artery was cannulated with polyethylene tubing (PE10). This catheter was attached to a pressure transducer that allowedinstantaneous measurement of mean arterial pressure (MAP) during theexperiment. For experiments using the pressure-controlled hemorrhagicshock (HS) model, the right jugular vein was exposed, ligated distally,and cannulated with polyethylene tubing (PE 10) in order to withdrawblood. For experiments using the volume-controlled hemorrhagic shock(HS) model, the jugular catheter was used to infuse the resuscitationsolution and the right femoral vein, which was cannulated with a siliconcatheter (CHRONIC-CATH, NORFOLK MEDICAL, Skokie, Ill.), was used towithdraw blood.

All animals were instrumented within 30 min. Heparin (500 U/kg) wasadministered immediately after instrumentation through the femoral vein.Animals were placed in a thermal blanket to maintain their bodytemperature at 37° C. The positioning of the different devicesaforementioned was checked postmortem.

Intestinal Mucosal Permeability Assay.

Animals were allowed access to water but not food for 24 h prior to theexperiment in order to decrease the volume of intestinal contents. Therats were instrumented as described above.

A midline laparotomy was performed and the small intestine wasexteriorized from the duodenojejunal junction to the ileocecal valve. Asmall incision was made on the antimesenteric aspect of the proximalsmall intestine and saline solution (1.5 ml) was injected. The bowel wasligated proximally and distally to the incision with 4-0 silk (LOOK,Reading, Pa.).

The small intestine was compressed gently in aboral direction along itslength to displace intestinal contents into the colon. Starting 5 cmfrom the ileocecal valve, the ileum was partitioned into six contiguouswater-tight segments. Each segment was 3 cm long and was boundedproximally and distally by constricting circumferential 4-0 silksutures. Care was taken to ensure that the vascular supply to intestinewas not compromised, and each segment was well-perfused.

Two randomly selected segments in each rat were injected with 0.3 ml ofvehicle and served as “no treatment” controls. In order to fill thesegments, a small incision was made and the solution was injected usinga Teflon catheter (ABBOCATH 16Ga, ABBOTT LABORATORIES).

The remaining four other segments were injected with solutionscontaining either 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl(TEMPOL) or one of the Gramicidin S-based compounds. Four differentfinal concentrations of TEMPOL in normal saline were evaluated: 0.1, 1,5 and 20 mM. The hemigramicidin-based compounds were dissolved in amixture of dimethylsulfoxide (DMSO) and normal saline (1:99 v/v) andinjected at final concentrations of 0.1, 1, 10 or 100 μM.

After the segments were loaded with saline or the test compounds, thebowel was replaced inside the peritoneal cavity and the abdominalincision was temporarily closed using Backhaus forceps.

After a 5 min stabilization period, hemorrhagic shock was induced bywithdrawing blood via the jugular catheter. MAP was maintained at 30±3mm Hg for 2 hours. The shed blood was re-infused as needed to maintainMAP within the desired range.

After 2 h of shock, the animals were euthanized with an intracardiac KClbolus injection. The ileum was rapidly excised from the ileocecal valveto the most proximal gut segment. The tips of each segment werediscarded. In order to assay caspases 3 and 7 activity and phospholipidsperoxidation, mucosa samples were collected from gut segmentsimmediately after hemorrhage and stored at −80° C. For permeabilitymeasurements, each segment was converted into an everted gut sac, aspreviously described by Wattanasirichaigoon et al., seeWattanasirichaigoon, S. et al., Effect of mesenteric ischemia andreperfusion or hemorrhagic shock on intestinal mucosal permeability andATP content in rats, SHOCK, 12:127-133 (1999).

Briefly, as per the Wattanasirichaigoon protocol referenced above, thesacs were prepared in ice-cold modified Krebs-Henseleit bicarbonatebuffer (“KHBB”), pH 7.4. One end of the gut segment was ligated with a4-0 silk suture; the segment was then everted onto a thin plastic rod.The resulting gut sac was mounted on a Teflon catheter (Abbocath 16GA,Abbot Laboratories) connected to a 3 ml plastic syringe containing 1.5ml of KHBB. The sac was suspended in a beaker containing KHBB plusfluorescein-isothiocyanate labeled dextran (average molecular mass 4kDa; FD4; 0.1 mg/ml). This solution was maintained at 37° C., andoxygenated by bubbling with a gas mixture (O₂ 95%/CO₂ 5%). After 30 min,the fluid within the gut sac was collected. The samples were cleared bycentrifugation at 2000 g for 5 min.

Fluorescence of FD4 in the solution inside the beaker and within eachgut sac was measured using a fluorescence spectrophotometer (LS-50,PERKIN-ELMER, Palo Alto, Calif.) at an excitation wavelength of 492 nmand an emission wavelength of 515 nm. Mucosal permeability was expressedas a clearance normalized by the length of the gut sac with units ofnL·min⁻¹·cm², as previously described, see Yang, R. et al., Ethylpyruvate modulates inflammatory gene expression in mice subjected tohemorrhagic shock, AM. J. PHYSIOL. GASTROINTEST. LIVER PHYSIOL.283:G212-G22 (2002).

Results for a specific experimental condition (i.e., specific testcompound at a single concentration) were expressed as relative change inpermeability calculated according to this equation: Relative change inpermeability (%)=(CH_(Hs exp)−C_(normal))/C_(Hs cont)−C_(normal))×100,where C_(Hs exp) is the clearance of FD4 measured for a gut segmentloaded with the experimental compound, C_(normal) is the clearance ofFD4 measured in 6 gut segments from 3 normal animals not subjected tohemorrhagic shock, and C_(Hs cont) is the mean clearance of FD4 measuredin 2 gut segments filled with vehicle from the same animal used tomeasure C_(HS exp).

Measurement of Permeability of Caco-2 Monolayers.

Caco-2_(BBe) cells were plated at a density of 5×10⁴ cells/well onpermeable filters (0.4 μm pore size) in 12-well bicameral chambers(TRANSWELL, COSTAR, Corning, N.Y.). After 21 to 24 days, paracellularpermeability was determined by measuring the apical-to-basolateralclearance of FD4.

Briefly, the medium on the basolateral side was replaced with controlmedium or medium containing menadione (50 μM final). Medium containingFD4 (25 mg/ml) was applied to the apical chamber. In some cases, one ofthe gramicidin S-based compounds, XJB-5-131, also was added to theapical side at final concentrations of 0.1, 1, 10 or 100 μM. After 6hours of incubation, the medium was aspirated from both compartments.Permeability of the monolayers was expressed as a clearance(pL·h⁻¹·cm⁻²), see Han, X. et al., Proinflammatory cytokines cause NOdependent and independent changes in expression and localization oftight junction proteins in intestinal epithelial cells, SHOCK 19:229-237(2003).

Caspases 3 and 7 Activity Assay.

Caspases 3 and 7 activity was measured using a commercially availableassay kit, CASPASE GLO™ 3/7 assay kit (PROMEGA, Madison, Wis.). Briefly,50 μl of rat gut mucosa homogenate (20 jug protein) was mixed with 50 μlof Caspase-G1oTM reagent and incubated at room temperature for 1 hour.At the end of incubation period, the luminescence of each sample wasmeasured using a plate reading chemiluminometer (ML1000, DYNATECHLABORATORIES, Horsham, Pa.). Activity of caspases 3 and 7 was expressedas luminescence intensity (arbitrary units per mg protein). Proteinconcentrations were determined using the BIORAD assay (BIO-RADLABORATORIES, Inc., Hercules, Calif.).

Assay for Peroxidation of Phospholipids.

Gut mucosal samples were homogenized. Lipids were extracted fromhomogenates using the Folch procedure, see M. Lees and G. H.Sloan-Stanley, A simple method for isolation and purification of totallipids from animal tissue, J. BIOL. CHEM. 226:497-509 (1957), andresolved by 2D HPTLC (High Performance Thin Layer Chromatography) aspreviously described, see Kagan, V. E. et al., A role for oxidativestress in apoptosis: Oxidation and externalization of phosphatidylserineis required for macrophage clearance of cell undergoing Fas-mediatedapoptosis, J. IMMUMOL. 169:487-489 (2002). Spots of phospholipids werescraped from HPTLC plates and phospholipids were extracted from silica.Lipid phosphorus was determined by a micro-method, see Bottcher, C. J.F. et al., A rapid and sensitive sub-micro phosphorus determination,ANAL. CHIM. ACTA 24: 203-204 (1961).

Oxidized phospholipids were hydrolyzed by pancreatic phospholipase A2 (2U/μl) in 25 mM phosphate buffer containing 1 mM CaCl₂, 0.5 mM EDTA and0.5 mM sodium dodecyl sulfate (SDS) (pH 8.0, at room temperature for 30min). Fatty acid hydroperoxides formed were determined by fluorescenceHPLC of resorufin stoichiometrically formed during their microperoxidase11-catalized reduction in presence of Amplex Red (for 40 min at 4° C.)(8). Fluorescence HPLC (ECLIPSE XDB-C18 column, 5 μm, 150×4.6 mm, mobilephase was composed of 25 mM disodium phosphate buffer (pH 7.0)/methanol(60:40 v/v); excitation wavelength 560 nm, emission wavelength 590 nm)was performed on a SHIMADZU LC-100AT HPLC system equipped withfluorescence detector (RF-10Ax1) and autosampler (SIL-10AD).

Survival of Rats Subjected to Volume-Controlled Hemorrhagic Shock.

Following surgical preparation and a 5-min stabilization period toobtain baseline readings, rats were subjected to hemorrhagic shock.Bleeding was carried out in 2 phases.

Initially, 21 ml/kg of blood was withdrawn over 20 min. Immediatelythereafter, an additional 12.5 ml/kg of blood was withdrawn over 40 min.Thus, hemorrhage occurred over a total period of 60 min and the totalblood loss was 33.5 ml/kg or approximately 55% of the total bloodvolume. Rats were randomly assigned to receive XJB-5-131 (2 μmol/kg) orits vehicle, a 33:67 (v/v) mixture of DMSO and normal saline. XJB-5-131solution or vehicle alone was administered as a continuous infusionduring the last 20 min of the hemorrhage period. The total volume offluid infused was 2.8 ml/kg and it was administered intravenously usinga syringe pump (KD100, KD SCIENTIFIC, New Hope, Pa.). Rats were observedfor 6 hours or until expiration (defined by apnea for >1 min). At theend of the 6 hour observation period, animals that were still alive wereeuthanized with an overdose of KCl.

Blood pressure was recorded continuously using a commercial strain-gaugetransducer, amplifier, and monitor (S90603a, SPACELABS, Redmond, Wash.).Blood samples (0.5 ml) were collected from the jugular vein at thebeginning of hemorrhage (baseline), at the end of hemorrhage (shock) andat the end of resuscitation (resuscitation). Hemoglobin concentration[Hb], lactate and glucose concentration were determined using anauto-analyzer (Model ABL 725, RADIOMETER COPENHAGEN, Westlake, Ohio).

Data Presentation and Statistics.

All variables are presented as means+Standard Error Mean (SEM).Statistical significance of differences among groups was determinedusing ANOVA (analysis of variance) and LSD (Least SignificantDifference) tests, or Kruskal-Wallis and Mann-Whitney tests asappropriate. Survival data were analyzed using the log-rank test.Significance was declared for p values less than 0.05.

Example I

Selective delivery of TEMPO to mitochondria could lead totherapeutically beneficial reduction of ROS; therefore, investigation ofthe use of conjugates of 4-amino-TEMPO (“4-AT”) was explored. In orderto selective target the mitochondria, a targeting sequence using themembrane active antibiotic Gramicidin S (“GS”) as well as correspondingalkene isosteres, shown in FIGS. 1 and 2. Accordingly, using theGramicidin S peptidyl fragments and alkene isosteres as “anchors,” theTEMPO “payload” could be guided into the mitochondria.

The Leu-^(D)Phe-Pro-Val-Orn fragment of Gramicidin S was used as atargeting sequence. Alkene isosteres such as (E)-alkene isosteres ofthis segment of Gramicidin S (i.e., hemigramicidin) were used as part ofthe targeting sequence. See FIG. 3 for the synthetic pathway for(E)-alkene isosteres and compound 3 for the corresponding chemicalstructure. The (E)-alkene as depicted in compound 2 of FIG. 2 was thenconverted in a multi-step process to yield the structure as depicted incompound 3 as an example of the (E)-alkene isostere.

Then, the compound depicted as compound 3 of FIG. 2 was conjugated withthe tripeptide H-Pro-Val-Orn(Cbz)-OMe using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (“EDC”) asa coupling agent. The tripeptide is an example of a suitable targetingsequence having affinity for the mitochondria of a cell. The resultingproduct is shown as compound 4a in FIG. 2. Saponification of compound 4afollowed by coupling with 4-amino-TEMPO (“4-AT”) afforded the resultingconjugates shown as compound 5a in FIG. 2, in which the Leu-^(D)Phepeptide bond has been replaced with an (E)-alkene.

In an alternate embodiment, conjugates 5b and 5c in FIG. 2 by couplingthe peptide 4b (Boc-Leu-^(D)Phe-Pro-Val-Orn(Cbz)-OMe) and the (E)-alkeneisostere as indicated as compound 3 in FIG. 2 to 4-AT. The peptide isanother example of a suitable targeting sequence having an affinity withthe mitochondria of a cell.

Electron paramagnetic resonance (“EPR”) spectroscopy was used to monitorthe cellular delivery of compounds 5a and 5b shown in FIG. 2 in mouseembryonic cells (“MEC”).

The following conditions were used during the EPR-based analysis of theintegration and reduction of nitroxide Gramicidin S-peptidyl conjugatesin MECs. The MECs at a concentration of 10 million MECs per mL wereincubated with 10 μM of 4-AT and compound 5a, respectively. Recoverednitroxide radicals in whole cells, mitochondria, and cytosol fractionswere resuspended in phosphate buffer saline (“PBS”) in the presence andabsence, respectively, of 2 μM K₃Fe(CN)₆. In brief, FIG. 3, panel Ashows a representative EPR spectra of compound 5a in different fractionsof MECs in the presence of K₃Fe(CN)₆. Further, FIG. 3, panel B shows anassessment of integrated nitroxides.

Distinctive characteristic triplet signals of nitroxide radicals weredetected in MECs incubated with 10 μM of compound 5a (FIG. 2) as well asin mitochondria isolated from these cells. The cytosolic function didnot elicit EPR signals of nitroxide radicals; similar results wereobserved with conjugate 5b (FIG. 2) (data not shown).

Incubation of MECs with compound 5a (FIG. 2) resulted in integration andone-electron reduction of compound 5a, as evidenced by a significantincrease in magnitude of the EPR signal intensity upon addition of aone-electron oxidant, ferricyanide (FIG. 3, panel B). (Note: EPR resultsfor incubation of MECs with 5b are not shown in FIG. 3; however, EPRresults for 5b were similar when compared to 5a). In contrast to 5a and5b, however, 4-amino-TEMPO (4-AT) did not effectively permeate cells orthe mitochondria, as shown by the absence of significant amplitudechange in the EPR results for 4-AT.

The ability of 5a, 5b (FIG. 2), and 4-AT to prevent intracellularsuperoxide generation by flow cytometric monitoring of oxidation ofdihydroehtidium (“DHE”) to a fluorescent ethidium was tested. Theability of 5a, 5b, and 4-AT to protect cells against apoptosis triggeredby actinomycin D (“ActD”) was also tested. MECs were pretreated with 10μM 4-AT, 5a, or 5b then incubated with ActD at a concentration of 100ng/mL. It was found that 5a and 5b completely inhibited nearly two-foldintracellular superoxide generation in MECs (see FIG. 5, panel A). 4-AThad no effect on the superoxide production in MECs.

Apoptotic cell responses were documented using three biomarkers: (1)externalization of phosphatidylserine (“PS”) on the cell surface (byflow cytometry using an FITC-labeled PS-binding protein, annexin V, seeFIG. 5, panels B and E); (2) activation of caspase-3 by cleavage of theZ-DEVD-AMC substrate (see FIG. 5, panel C), and, (3) DNA fragmentationby flow cytometry of propidiium iodide stained DNA (see FIG. 5, panelD).

Phosphatidylserine (“PS”) is an acidic phospholipid located exclusivelyon the inner leaflet of the plasma membrane; exposure of PS on the cellsurface is characteristic of cell apoptosis. Externalization of PS wasanalyzed by flow cytometry using an annexin V kit. Cells were harvestedby trypsinization at the end of incubation and then stained with annexinV-FITC and propidium iodide (“PS”). Ten thousand cell events werecollected on a FACScan flow cytometer. Cells that annexin V-positive andPI-negative were considered apoptotic.

Activation of capase-3, a cystein protease only activated in theexecution phase of apoptosis, was determined using an EnzChek capsase-3assay kit.

Further, calcium and magnesium dependent nucleases are activated thatdegrade DNA during apoptosis. These DNA fragments are eluted, stainedwith propidium iodide and analyzed using flow cytometry. A cellpopulation with decreased DNA content was considered a fraction ofapoptotic cells.

Anti-apoptotic effects of compounds 5a and 5b were observed atrelatively low concentrations of 10 μM. Compounds 5a and 5b (FIG. 3)reduced the number of annexin V-positive cells as shown in FIG. 5, panelB, prevented caspase-3 activation as shown in FIG. 5, panel C, andprevented DNA fragmentation as shown in FIG. 5, panel D. Atconcentrations in excess of 10 μM, both 5a and 5b were either lessprotective or exhibited cytotoxicity (FIG. 5, panel E). In contrast,4-AT afforded no protection.

In contrast, compound 5c, which does not have a complete targetingmoiety, was ineffective in protecting MECs against ActD-inducedapoptosis (FIG. 5, panels B and C) at low concentrations. Accordingly,the hemigramicidin peptidyl targeting sequence is essential foranti-apoptotic activity of nitroxide conjugates such as those containingTEMPO.

Finally, the reduction of compounds 5a and 5b could also causeinhibition of mitochondrial oxidative phosphorylation, so the ATP levelsof MECs treated with these compounds were tested. As is known to oneordinarily skilled in the art, ATP serves as the primary energy sourcein biological organisms; reduction of ATP levels would greatly impairnormal cell function. ATP levels in MECs in the presence or absence of5a or 2-deoxyglucose (“2-DG”) were used as a positive control (see FIG.5, panel F). At concentrations at which anti-apoptotic effects weremaximal (˜10 μM, FIG. 5, panel E), nitroxide conjugates did not causesignificant changes in the cellular ATP level. Therefore, syntheticGS-peptidyl conjugates migrate into cells and mitochondria where theyare reduced without affecting the ability of the mitochondria to produceATP.

Example II

In an in vivo assay, the ileum of rats was divided into a series ofwell-vascularized components in a manner akin to links of sausage. Thelumen of each ileal compartment was filled with a 3 μL aliquot of testsolution. Two of the ileal compartments were filled with vehicle alone(i.e., a solution containing at least in part the TEMPO derivative).These two components served as internal controls to account forindividualistic variations in the severity of shock or the response ofthe mucosa to the shock.

Using this assay system, eight compounds were evaluated as shown in FIG.5: TEMPOL (FIG. 4A, panel A), one dipeptidic TEMPO analog (FIG. 4A,panel B—XJB-5-208), 3 hemigramicidin-TEMPO conjugates (FIG. 4B, panel CXJB-5-125, 4C, panel E XJB-5-131, and 4D, panel G XJB-5-197), and 3hemigramicidin compounds that do not have the TEMPO moiety (FIG. 4B,panel D—XJB-5-127, 4C, panel F—XJB-5-133, and 4D, panel H-XJB-5-194).

Hemorrhagic shock in rats leads to marked derangements in intestinalmucosal barrier function—in other words, the mucosal permeability ofshocked intestinal segments was significantly greater than thepermeability of segments from normal rats (52.3+0.5 versus 6.9+0.1nL·min⁻¹·cm⁻², respectively; p<0.01), see Tuominen, E. K. J.,Phospholipid cytochrome c interaction: evidence for the extended lipidanchorage, J. BIOL. CHEM., 277:8822-8826 (2002); also Wipf P. et al.,Mitochondria targeting of selective electron scavengers: synthesis andbiological analysis of hemigramicidin-TEMPO conjugates, J. AM. CHEM.SOC. 127:12460-12461. Accordingly, mice were subjected to 2 hours ofshock (Mean Arterial Pressure (“MAP”)=30 f 3 mm Hg), the gut segmentswere harvested and mucosal permeability to flouresceinisothiocyanate-dextran (“FD4”) measured ex vivo. Data in FIG. 4 areexpressed as a percentage of the change permeability relative to thatobserved in simultaneously assayed control segments loaded during shockwith normal saline solution.

Accordingly, intraluminal TEMPOL was used as a “positive control” forgut mucosal protection assay. TEMPOL concentrations >1 mM in the gutlumen ameliorated hemorrhagic shock-induced ileal mucosalhyperpermeability (FIG. 5, panel A). Two of the TEMPO conjugates, namelyXJB-5-208 (FIG. 4A, panel B) and XJB-5-131 (FIG. 4B, panel C), alsosignificantly ameliorated hemorrhagic shock-induced ileal mucosalhyperpermeability. The lowest effective concentration for XJB-5-208(FIG. 4A, panel B) and XJB-5-131 (FIG. 4C, panel E) was 1 μM; i.e., bothof these compounds were ˜1000-fold more potent than TEMPOL. Two othercompounds carrying the TEMPO payload, XJB-5-125 (FIG. 4B, panel C) andXJB-5-197 (FIG. 4D, panel G) failed to provide protection against gutbarrier dysfunction induced by hemorrhage. XJB-5-133 (FIG. 4C, panel F)has the same (hemigramicidin-based) mitochondrial targeting moiety asXJB-5-131 (FIG. 4B, panel E) but lacks the TEMPO payload. It isnoteworthy, therefore, that XJB-5-133 (FIG. 4C, panel F) did not affordprotection from the development of ileal mucosal hyperpermeability.

Ineffective as well were the two other hemigramicidin-based compoundsthat also lacked the TEMPO payload, XJB-5-127 (FIG. 4B, panel D) andXJB-5-194 (FIG. 4D, panel H). Of the compounds screened, XJB-5-131 (FIG.4C, panel E) appeared to be the most effective, reducing hemorrhagicshock-induced mucosal hyperpermeability to approximately 60% of thecontrol value.

Based upon the results as reflected in FIG. 4A-4D, both the TEMPOpayload and the “anchoring” hemigramicidin fragment are requisitemoieties that should be present in order for effective electronscavenging activity by the XJB-5-131 compound. Accordingly, it was foundthat XJB-5-131 ameliorates peroxidation of mitochondrial phosopholipids(i.e., ROS activity) in gut mucosa from rats subject to hemorrhagicshock.

In the subsequent series of in vivo studies, the affect of intraluminalXJB-5-131 on hemorrhage-induced peroxidation of phospholipids inintestinal mucosa was examined. Isolated segments of the ileum of ratswere divided into a series of well-vascularized components in a mannerakin to sausage and the lumen of each ileal compartment was filled withthe same volume of test solution containing either vehicle or a 10 μMsolution of XJB-5-131, which was previously indicated to be the mostactive of the hemigramicidin-TEMPO conjugates. In a preferredembodiment, 0.3 mL of test solution filled the lumen of each ilealcompartment.

After two hours of HS, samples of ileal mucosa from the gut sacs filledwith the vehicle and XJB-5-131 were obtained and compared with ilealmucosa of normal MECs. All samples were assayed with caspase 3 orcaspase 7 activity as well as the peroxidation of phosphatidylcholine(“PC”), phosphatidylethanolamine (“PE”), phosphatidylserine (“PS”), andcardiolipin (“CL”), summarized in FIG. 6.

As can be seen in FIG. 6, treatment with XJB-5-131 significantlyameliorated hemorrhage-induced peroxidation of CL, the only phospholipidtested found in mitochondria. However, treatment with XJB-5-131 only hada small effect on PE peroxidation and no effect on peroxidation of PCand PS. Based upon these trends, hemorrhagic shock is associated withsubstantial oxidative stress even in the absence of resuscitation.Further, this data also establishes that XJB-5-131 is an effective ROSscavenger as it localizes predominantly in mitochondria and protects CLfrom peroxidation.

Relative to the activity measured in samples from normal animals, theactivity of caspases 3 and 7 was markedly increased in vehicle-treatedmucosal samples from hemorrhaged rats (FIG. 7). However, when the ilealsegments were filled with XJB-5-131 solution instead of its vehicle, thelevel of caspase 3 and 7 activity after hemorrhagic shock wassignificantly decreased. Accordingly, hemorrhagic shock is associatedwith activation of pro-apoptotic pathways in gut mucosal cells.Moreover, the data support the view that this process is significantlyameliorated following mitochondrial treatment with XJB-5-131.

Example III

In another series of experiments, monolayers of enterocyte-like cells,Caco-2_(BBe), were studied for physiological and pathophysiologicalpurposes for determining intestinal barrier function. Just as with theprior Example I and II with respect to ROS exposure, the permeability ofCaco-2_(BBe) monolayers increases when the cells are incubated with theROS, hydrogen peroxide, or menadione (a redox-cycling quinine thatpromotes the formation of superoxide anion radicals), see Baker, R. D.et al., Polarized Caco-2 cells, Effect of reactive oxygen metabolites onenterocyte barrier function, DIGESTIVE DIS. SCI. 40:510-518 (1995); alsoBanan, A. et al., Activation of delta-isoform of protein kinase C isrequired for oxidant-induced disruption of both the microtubulecytoskeleton and permeability barrier of intestinal epithelia, J.PHARMACOL. EXP. THER. 303:17-28 (2002).

Due to the results with respect to XJB-5-131 and its amelioration ofhemorrhage-induced CL peroxidation in mucosal cells in vivo (seeaforementioned Example I and II), a possible treatment using XJB-5-131was investigated to determine if menadione-induced epithelialhyperpermeability could be ameliorated in vitro. Consistent with theprior in vivo observations, Caco-2_(BBe) monolayers were incubated inthe absence and in the presence of menadione, respectively. After 6hours, incubation of Caco-2_(BBe) monolayers with menadione caused amarked increase in the apical-basolateral clearance of FD4 (FIG. 8).Treatment with 10 μM XJB-5-131 provided significant protection againstmenadione-induced hyperpermeability.

Example IV

As reflected by the above in vivo and in vitro studies, XJB-5-131 hadsignificantly beneficial effects on several biochemical andphysiological read-outs. Accordingly, systemic administration ofXJB-5-131 was investigated with respect to whether it would prolongsurvival of patients subjected to profound periods of hemorrhagic shockwith massive blood loss in the absence of standard resuscitation withblood and crystalloid solution. As in the above studies, rats wereutilized as test patients.

A total of sixteen rats were tested in this study. Rats were treatedwith 2.8 ml/kg of vehicle or the same volume of XJB-5-131 solutionduring the final 20 min of the bleeding protocol. The total dose ofXJB-5-131 infused was 2 μmol/kg. Following profound hemorrhagic shockconsistent with the protocol described above for the prior studies,thirteen survived for at least 60 min and received the full dose ofeither XJB-5-131 solution or the vehicle, a 33:67 (v/v) mixture of DMSOand normal saline. As shown in Table 1, blood glucose, lactate andhemoglobin concentrations were similar in both groups at baseline andbefore and immediately after treatment. None of the between-groupdifferences were statistically significant.

TABLE 1 End of End of first second phase of phase of Parameter CompoundBaseline hemorrhage hemorrhage Blood glucose Vehicle 143 ± 5  255 ± 30219 ± 26  concentration XJB-5-131 134 ± 4  228 ± 24 201 ± 38  (mg/dL)Blood lactate Vehicle 1.8 ± 0.4  606 ± 0.8 5.9 ± 1.3 concentrationXJB-5-131 1.8 ± 0.2  5.7 ± 0.8 5.6 ± 1.2 (mEq/L) Blood Hb Vehicle 12.7 ±0.5  11.1 ± 0.3 9.4 ± 0.2 concentration XJB-5-131 12.7 ± 0.3  10.7 ± 0.39.4 ± 0.3 (g/dL)

Shortly after treatment was started, mean arterial pressure (“MAP”)increased slightly in both groups (see FIG. 9A). In both groups, meanarterial pressure (“MAP”) decreased precipitously during the first phaseof the hemorrhage protocol and remained nearly constant at 40 mm Hgduring the beginning of the second phase. Six of the seven animals inthe vehicle-treated (control) group died within one hour of the end ofthe bleeding protocol and all were dead within 125 minutes (FIG. 9B).Rats treated with intravenous XJB-5-131 survived significantly longerthan those treated with the vehicle. Three of the six rats survivedlonger than 3 hours after completion of the hemorrhage protocol; one ratsurvived the whole 6 hour post-bleeding observation period (FIG. 9B).

Analysis

Accordingly, analysis of the XJB-5-131 studies indicate that exposure ofthe patient to the compound prolongs the period of time that patientscan survive after losing large quantities of blood due to traumaticinjuries or other catastrophes (e.g., rupture of an abdominal aorticaneurysm).

By extending the treatment window before irreversible shock develops,treatment in the field with XJB-5-131 might “buy” enough time to allowtransport of more badly injured patients to locations where definitivecare, including control of bleeding and resuscitation with bloodproducts and non-sanguineous fluids, can be provided. The results usinga rodent model of hemorrhagic shock also open up the possibility thatdrugs like XJB-5-131 might be beneficial in other conditions associatedwith marked tissue hypoperfusion, such as stroke and myocardialinfarction.

The results presented here also support the general concept thatmitochondrial targeting of ROS scavengers is a reasonable therapeuticstrategy. Although previous studies have shown that treatment withTEMPOL is beneficial in rodent HS situations, a relatively large dose ofthe compound was required (30 mg/kg bolus+30 mg/kg per h). In contrast,treatment with a dose of XJB-5-131 that was about 300 fold smaller (˜0.1mg/kg) was clearly beneficial. The greater potency of XJB-5-131 ascompared to TEMPOL presumably reflects the tendency of XJB-5-131 tolocalize in mitochondrial membranes, a key embodiment of the invention.As indicated in Example I, two hemigramicidin-4-amino-TEMPO conjugates(namely XJB-5-208 and XJB-5-131, see FIG. 2) are concentrated in themitochondria of cultures mouse embryonic cells following incubation withsolutions of the compounds.

Further, the use of XJB-5-131 significantly prolonged the survival ofthe rats subjected to massive blood loss, even though the animals werenot resuscitated with either blood or other non-sanguineous fluids andthey remained profoundly hypotensive.

In light of the above, synthetic hemigramicidin peptidyl-TEMPOconjugates permeate through the cell membrane and also the mitochondrialmembrane where they act as free radical scavengers for ROS such as, butnot limited to, superoxide anion radicals. The conjugates are thenreduced within the mitochondria by electron-transport proteins which areinvolved with the cellular respiration pathway, thereby coupling thedecoupled ROS species. These conjugates also have the advantage, asdiscussed above, of being anti-apoptotic, especially in the case ofcompounds such as 5a and 5b.

By effectively reducing the amount of ROS species, a patient'scondition, including an illness or other medical condition, may beameliorated and, in some cases, survival may be prolonged as describedin the Example IV study. Examples of such conditions, including diseasesand other medical conditions, include (but are not limited to) thefollowing medical conditions which include diseases and conditions:myocardial ischemia and reperfusion (e.g., after angioplasty andstenting for management of unstable angina or myocardial infarction),solid organ (lung liver, kidney, pancreas, intestine, heart)transplantation, hemorrhagic shock, septic shock, stroke, tissue damagedue to ionizing radiation, lung injury, acute respiratory distresssyndrome (ARDS), necrotizing pancreatitis, and necrotizingenterocolitis.

Example V

In a further embodiment of the invention, a composition for scavengingradicals in a mitochondrial membrane comprises a radical scavengingagent or an NOS inhibitor and a membrane active peptidyl fragment havinga high affinity with the mitochondrial membrane. The membrane activepeptidyl fragment preferably has a property selected from the groupconsisting of antioxidant, radioprotective, protective, anti-apoptotic,therapeutic, ameliorative, NOS antagonist and combinations thereof.

In a related embodiment, with respect to compounds with antibioticproperties, it is generally preferable to employ compounds whose mode ofaction includes bacterial wall targets.

In a preferred embodiment, the membrane active compound is preferablyselected from the group consisting of bacitracins, gramicidins,valinomycins, enniatins, alamethicins, beauvericin, serratomolide,sporidesmolide, tyrocidins, polymyxins, monamycins, and lissoclinumpeptides.

In a related embodiment, the NOS antagonist is selected from the groupconsisting of XJB-5-234 (a), XJB-5-133 (b), XJB-5-241 (c), and XJB-5-127(d):

Example VI

The following examples provide protocols for additional cargo usable inthe present invention for compounds which serve as NOS antagonists.

Compound (1) is Boc-Leu-ψ[(E)-C(CH₃)═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-AMT(XJB-5-241) and was prepared according to the following protocol. Asolution of 11.0 mg (13.2 μmol) ofBoc-Leu-ψ[(E)-C(CH₃)═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-OMe (2-48) in 400 μLof MeOH was treated at 0° C. with 132 μL (132 μmol) of 1 N NaOH. Thereaction mixture was stirred at room temperature for 8 h, and treatedwith 132 μL (132 μmol) of 1 N HCl. The solution was extracted with CHCl₃and the organic layer was dried (Na₂SO₄) and concentrated in vacuo togive the crude acid as a colorless form. This acid was dissolved in 2.00mL of CHCl₃ and treated at room temperature with 2.1 mg (16 μmol) ofHOBt, 3.0 mg (16 μmol) of EDC, 3.3 mg (20 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 3.5 mg (27 μmol) ofDMAP. The reaction mixture was stirred at room temperature for 48 h,diluted with CHCl₃, and washed with H₂O. The organic layer was dried(Na₂SO₄), concentrated in vacuo, and purified by chromatography on SiO₂(1:1, hexanes/EtOAc followed by 20:1, CHCl₃/MeOH) to yield 11 mg (89%)of XJB-5-241 as a colorless powder. The following characterization datawere obtained: LC-MS (R_(t) 8.37 min, linear gradient 70% to 95% CH₃CN(H₂O) in 10 min, 0.4 mL/min; m/z=932.4 [M+H]⁺, 954.3 [M+Na]⁺) and HRMS(ESI) m/z calculated for C₅₀H₇₄N₇O₈S (M+H) 932.5320. found 932.5318.

Compound (2) is Boc-Leu-ψ[(E)-CH═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-AMT(XJB-5-133) and was prepared according to the following protocol. Asolution of 20.0 mg (24.3 μmol) of 2-85 (XJB-5-194) in 800 μL of MeOHwas treated at 0° C. with 243 μL (243 μmol) of 1 N NaOH. The reactionmixture was stirred at room temperature for 6 h, and treated with 243 μL(243 μmol) of 1 N HCl. The solution was extracted with CHCl₃ and theorganic layer was dried (Na₂SO₄) and concentrated in vacuo to give thecrude acid as a colorless form. This acid was dissolved in 1.00 mL ofCHCl₃ and treated at room temperature with 3.9 mg (29 μmol) of HOBt, 5.6mg (29 μmol) of EDC, 6.1 mg (37 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 7.4 mg (61 μmol) ofDMAP. The reaction mixture was stirred at room temperature for 20 h,diluted with CHCl₃, and washed with H₂O. The organic layer was dried(Na₂SO₄), concentrated in vacuo, and purified by chromatography on SiO₂(1:1, hexanes/EtOAc followed by 20:1, CHCl₃/MeOH) and an additionalpreparative C18 reverse phase HPLC purification was performed: 80% to100% CH₃CN (H₂O) in 20 min, 5.0 mL/min) to afford 12.9 mg (58%) ofXJB-5-133 as a colorless powder. The following characterization datawere obtained: LC-MS(R_(t) 7.89 min, linear gradient 70% to 95% CH₃CN(H₂O) in 10 min, 0.4 mL/min; m/z=918.3 [M+H]⁺, 940.3 [M+Na]⁺) and HRMS(ESI) m/z calculated for C₄₉H₇₂N₇O₈S (M+H) 918.5163. found 918.5185.

Compound (3) is Boc-Leu-^(D)Phe-Pro-Val-Orn(Cbz)-AMT (XJB-5-127).According to the following protocol. A solution of 24.0 mg (28.7 μmol)of Boc-Leu-^(D)Phe-Pro-Val-Orn(Cbz)-OMe in 800 μL of MeOH was treated atroom temperature with 287 μL (287 μmol) of 1 N NaOH. The reactionmixture was stirred at room temperature for 5 h, and treated at 0° C.with 287 μL (287 μmol) of 1 N HCl. The solution was extracted with CHCl₃and the organic layer was dried (Na₂SO₄) and concentrated in vacuo togive the crude acid as a colorless foam. The crude acid was dissolved in2.00 mL of CHCl₃ and treated at room temperature with 4.6 mg (34 μmol)of HOBt, 6.6 mg (34 μmol) of EDC, 5.7 mg (34 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 8.8 mg (72.0 μmol)of DMAP. The reaction mixture was stirred at room temperature for 24 h,diluted with CHCl₃, and washed with H₂O. The organic layer was dried(Na₂SO₄), concentrated in vacuo, and purified by chromatography on SiO₂(2:1, hexanes/EtOAc followed by 20:1, CHCl₃/MeOH) to yield 17.0 mg (63%)of XJB-5-127 as a colorless powder. The following characterization datawere obtained: LC-MS(R_(t) 6.32 min, linear gradient 70% to 95% CH₃CN(H₂O) in 10 min, 0.4 mL/min; m/z=935.3 [M+H]⁺, 957.3 [M+Na]⁺) and HRMS(ESI) m/z calculated for C₄₈H₇₁N₈O₉S (M+H) 935.5065. found 935.5044.

Compound (4) is Boc-Leu-ψ[(E)-CH═CH]-^(D)Phe-AMT (XJB-5-234). A solutionof crude Boc-Leu-ψ[(E)-CH═CH]-^(D)Phe-OH (2-84) (30.5 μmol) in 2.00 mLof CH₂Cl₂ was treated at 0° C. with 6.1 mg (37 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl, 7.0 mg (37 μmol) ofEDC, 4.9 mg (37 μmol) of HOBt, and 9.3 mg (76 μmol) of DMAP. Thereaction mixture was stirred at room temperature overnight, concentratedin vacuo, and purified by chromatography on SiO₂ (2:1, CH₂Cl₂/EtOAc) toyield 9.1 mg (63%) of XJB-5-234 as a colorless foam. The followingcharacterization data were obtained: LC-MS(R_(t) 8.42 min, lineargradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=474.5 [M+H]⁺)and HRMS (ESI) m/z calculated for C₂₆H₄₀N₃O₃S (M+H) 474.2790. found474.2781.

Compound (5) is Boc-Leu-ψ[(Z)—CF═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-TEMPO(XJB-7-53). A solution of 3.4 mg (4.1 μmol) ofBoc-Leu-ψ[(Z)—CF═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-OMe XJB-5-66) in 400 μL ofMeOH was treated at 0° C. with 41 μL (41 μmol) of 1 N NaOH. The reactionmixture was stirred at room temperature for 12 h, and treated with 41 μL(41 μmol) of 1 N HCl. The solution was extracted with CHCl₃ and theorganic layer was dried (Na₂SO₄) and concentrated in vacuo to give thecrude acid as a colorless form. This acid was dissolved in 400 μL ofCHCl₃ and treated at room temperature with 0.7 mg (5 μmol) of HOBt, 0.9mg (5 μmol) of EDC, 0.5 mg (4 μmol) of 4-amino-TEMPO and 1.1 mg (6 μmol)of DMAP. The reaction mixture was stirred at room temperature for 12 h,diluted with CHCl₃, and washed with H₂O. The organic layer was dried(Na₂SO₄), concentrated in vacuo, and purified by chromatography on SiO₂(1:1, hexanes/EtOAc followed by 20:1, CHCl₃/MeOH) to yield 3.6 mg (91%)of XJB-7-53 as a colorless powder. The following characterization datawere obtained: LC-MS(R_(t) 8.45 min, linear gradient 70% to 95% CH₃CN(H₂O) in 10 min, 0.4 mL/min; m/z=977.5 [M+H]⁺, 999.5 [M+Na]⁺) and HRMS

(ESI) m/z calculated for C₅₃H₇₉FN₇O₉Na (M+Na) 999.5821.

Compound (6) is Boc-^(D)Phe-ψ[(E)-C(CH₃)═CH]-Ala-Val-Orn(Cbz)-Leu-AMT(XJB-7-42) and was prepared according to the following protocol. Asolution of 4.5 mg (5.6 μmol) ofBoc-^(D)Phe-ψ[(E)-C(CH₃)═CH]-Ala-Val-Orn(Cbz)-Leu-OMe (2-119) in 0.35 mLof MeOH was treated at 0° C. with 56 μL (56 μmol) of 1 N NaOH. Thereaction mixture was stirred at room temperature for 12 h, and treatedwith 56 μL (56 μmol) of 1 N HCl. The solution was extracted with CHCl₃and the organic layer was dried (Na₂SO₄) and concentrated in vacuo togive the crude acid as a colorless form. This acid was dissolved in 0.80mL of CHCl₃ and treated at room temperature with 0.9 mg (6.7 μmol) ofHOBt, 1.3 mg (6.7 μmol) of EDC, 1.4 mg (8.4 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 1.7 mg (14 μmol) ofDMAP. The reaction mixture was stirred at room temperature for 36 h,concentrated in vacuo, and purified by chromatography on SiO₂ (20:1,CHCl₃/MeOH) to yield 5.0 mg (99%) of XJB-7-42 as a colorless foam. Thefollowing characterization data were obtained: LC-MS(R_(t) 6.61 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=907.3[M+H]⁺, 929.4 [M+Na]⁺) and HRMS (ESI) m/z calculated for C₄₈H₇₂N₇O₈S(M+H) 906.5163. found 906.5190.

Compound (7) is Boc-^(D)Phe-ψ[(E)-C(CH₃)═CH]-Ala-Val-AMT (XJB-7-43). Asolution of 14.3 μmol of crude Boc-^(D)Phe-ψ[(E)-C(CH₃)═CH]-Ala-Val-OMe(2-111) in 1.00 mL of CHCl₃ was treated at room temperature with 2.3 mg(17 μmol) of HOBt, 3.3 mg (17 μmol) of EDC, 3.6 mg (22 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 4.4 mg (36 μmol) ofDMAP. The reaction mixture was stirred at room temperature for 36 h,concentrated in vacuo, and purified by chromatography on SiO₂ (20:1,CHCl₃/MeOH) to yield 7.5 mg (96%) of XJB-7-43 as a colorless foam. Thefollowing characterization data were obtained: LC-MS(R_(t) 5.41 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=545.3[M+H]⁺, 567.3 [M+Na]⁺) and HRMS (ESI) m/z calculated for C₂₉H₄₄N₄O₄S(M+Na) 567.2981. found 567.2971.

Among the preferred radical scavenging agents are a material selectedfrom the group consisting of a ubiquinone analog, a ubiquinone analogfragment moiety, a ubiquinone analog fragment moiety lacking ahydrophilic tail, a superoxide dismutase mimetic, a superoxide dismutasebiomimetic or a salen-manganese compound.

As is known to one ordinarily skilled in the art, ionizing radiationactivates a mitochondrial nitric oxide synthase (“mtNOS”), leading toinhibition of the respiratory chain, generation of excess superoxideradicals, peroxynitrite production and nitrosative damage. The damagedone by ionizing radiation is believed to be alleviated [See Kanai, A.J. et al., AMERICAN JOURNAL OF PHYSIOLOGY 383: F1304-F1312 (2002); andKanai, A. J. et al., AMERICAN JOURNAL OF PHYSIOLOGY 286: H13-H21(2004)]. The composition of this embodiment is characterized by theproperty of inhibiting mtNOS, thereby resisting generation of excesssuperoxide radicals, peroxynitrite and nitrosative damage.

Protection again irradiation damage using systemic drug delivery canresult in unwanted side effects. One approach to limit or prevent theseadverse side affects is to target drug delivery to the mitochondriausing a peptide carrier strategy.

The present invention may employ a Gramicidin-S fragment moiety that canbe varied in length and the Orn side chain amines can be acylated tomodulate passage through the cell membrane and mitochondrial targeting.

In a preferred embodiment of the invention, a potent NOS inhibitor, thenon-arginine analog of 2-amino-6-methyl-thiazine (“AMT”), was selected.Irradiation of the ureopithelium results in increased production ofsuperoxide and nitric oxide (“NO”), mouse bladders were instilled withAMT or 4-amino-TEMPO to determine if inhibition of NO or scavenging freeradicals is more radioprotective.

An unconjugated and conjugated NOS antagonist, (AMT, 100 μM) and anunconjugated and conjugated nitroxide derivative (4-amino-TEMPO, 100 μM)were incubated for two hours at 37° C. with 32D c13 hemopoietic cells.

Following incubation, the cells were lysed and the mitochondria isolatedfor a mass spectrometry analysis where compounds isolated frommitochondria were identified as Na+ adducts. The resulting spectra (notshown) demonstrate that 4-amino-TEMPO only permeate the mitochondrialmembrane with the assistance of the attached GS-derived targetingsequence. Further spectra (not shown) indicate that unconjugated AMT donot enter the mitochondria membrane in substantial quantities. Thus, thetargeting peptides successfully direct a NOS antagonist and a nitroxideto the mitochondria.

Further physiological studies were conducted to determine the effects ofpeptide-targeted AMT and 4-amino-TEMPO on NO and peroxynitriteproduction in irradiated uroepithelial cells. The cells were cultured inan 8-well slide chamber for 3 days and then microsensor measurementswere taken 24 hours after irradiation.

In untreated irradiated cells and cells treated with unconjugated4-amino-TEMPO (100 μM) or unconjugated AMT (10 μM), capsaicin evoked NOproduction and resulted in the formation of comparable amount ofperoxynitrite. In cells treated with high-dose conjugated 4-amin-TEMPO(100 μM), peroxynitrite production was decreased by approximately4-fold. In non-radiated cells or cells treated with conjugated AMT (10μM), NO induced peroxynitrite formation was nearly completely inhibited.This suggested that peptides conjugates couple or covalently link withmembrane impermeant 4-amino-TEMPO or AMT and facilitate the transport of4-amin-TEMPO across the mitochondrial membrane. Furthermore, this datasuggests that the peptide conjugates do not interfere with the NOSinhibitory activity of AMT or the free radical scavenging activity of4-amino-TEMPO and that AMT is a more effective radioprotectant [Kanai,A. J. et al., Mitochondrial Targeting of Radioprotectants Using PeptidylConjugates, ORGANIC AND BIOMOLECULAR CHEMISTRY (in press)].

Quantitative mass spectrometry studies were used to compare theeffectiveness of several AMT peptide conjugates in permeating themitochondrial membrane, specifically XJB-5-234, XJB-5-133, XJB-5-241,and XJB-5-127. The Fmole/10 μM mitochondrial protein ratio provides arelative quantification of conjugate concentration at the target site.Table 2 indicates that the most efficacious conjugate was compoundXJB-5-241.

TABLE 2 Compound Fmole/l0M mitochondrial protein XJB-5-234 1.45XJB-5-133 89.8 XJB-5-241 103.3

The trisubstituted (E)-alkene moiety embedded in XJB-5-241 has astronger conformational effect that the less biologically activedisubstituted (E)-alkene XJB-5-133 or the GS peptidyl fragmentXJB-5-127, see Wipf P. et al., Methyl-and (Triluoromethyl)alkene PeptideIsosteres: Synthesis and Evaluation of Their Potential as β-TurnPromoters and Peptide Mimetics J ORG. CHEM. 63:6088-6089 (1998); alsoWipf P. et al., Imine Additions of Internal Alkynes for the Synthesis ofTrisubstituted (E)-Alkene and Cyclopropane Peptide Isosteres ADV. SYNTH.CAT. 347:1605-1613 (2005). The data indicates that a defined secondarystructure and an appropriate conformational preorganization is importantin accomplishing mitochondrial permeation of compounds that reducenitrosative and oxidative effects.

The presence of a non-hydrolyzable alkene isostere functions in place oflabile peptide bonds and is significant for a prolonged mechanism ofaction. The relatively rigid (E)-alkenes (ψ[(E)-C(R)═CH]) representuseful, conformationally preorganized structural mimetics and have beenused as surrogates of hydrolytically labile amide bonds in a number ofenzyme inhibitors. The primary objective of this strategy is theaccurate mimicry of the geometry of the peptide bond; however,(E)-alkenes also modulate the physicochemical properties, solubility,and lipophilicity, number of hydrogen donors and acceptors, etc, of theparent structures, and therefore generally have a different metabolicfate than simple peptides.

A targeted delivery strategy employed in this invention is advantageoussince some neuronal NOS (nNOS) antagonists and most antioxidants,including nitroxide derivatives, are poorly cell-permeable and requiretherapeutically effective concentrations greater than 100 μM if usedwithout a conjugate.

The method related to this embodiment of the invention delivers acomposition to mitochondria comprising transporting to said mitochondriaa desired cargo which may, for example, be (a) a radical scavengingagent by use of a membrane active peptidyl fragment preferably havinghas a β-turn motif having a high affinity for the mitochondrial membraneor (b) a nitric oxide synthase antagonist bonded to the membrane activepeptidyl fragment.

Whereas particular embodiments of this invention have been describedabove for purposes of illustration, it will be evident to those skilledin the art that numerous variations of the details of the presentinvention may be made without departing from the invention as defined inthe appended claims.

1-77. (canceled)
 78. A method of delivering a cargo to a mitochondria ofa patient comprising administering to a patient a mitochondria-targetingcompound comprising a membrane-active fragment of gramicidin S or anE-alkylene isostere thereof conjugated to a cargo.
 79. The method ofclaim 78, in which the gramicidin S fragment or E-alkylene isosterethereof comprises a β-turn.
 80. The method of claim 78, in which thegramicidin S fragment or E-alkylene isostere thereof has the sequenceLeu-^(D)Phe-Pro-Val-Orn.
 81. The method of claim 78, in which thegramicidin S fragment or E-alkylene isostere thereof is an E-alkyleneisostere having the sequence Leu-(E)-^(D)Phe-Pro-Val-Orn.
 82. The methodof claim 78, in which an amine of the gramicidin S fragment orE-alkylene isostere thereof is acylated.
 83. The method of claim 78, inwhich an amine of the gramicidin S fragment or E-alkylene isosterethereof is acylated with Boc and/or Cbz.
 84. The method of claim 78, inwhich the cargo is a reactive-oxygen species (ROS) scavenger or an NOSinhibitor.
 85. The method of claim 84, in which the cargo is a nitroxidereactive-oxygen species (ROS) scavenger ROS scavenger.
 86. The method ofclaim 78, in which the in which the cargo is TEMPO or 4-amino TEMPO. 87.The method of claim 78, in which the cargo is conjugated to the compoundof formula A:

where R is H or methyl.
 88. The method of claim 87, in which the cargois TEMPO or 4-amino TEMPO.
 89. The method of claim 78, in which the ROSis conjugated to the compound of Formula B:


90. The method of claim 89, in which the cargo is TEMPO or 4-aminoTEMPO.
 91. The method of claim 78, in which the compound is XJB-5-131.92. The method of claim 78, in which the compound is XJB-5-208.
 93. Themethod of claim 78, in which the compound is administered to treat apatient for a condition associated or caused by reactive oxygen species(ROS), and the cargo is an ROS scavenger.
 94. The method of claim 93, inwhich the condition is hemorrhagic shock.
 95. A compound comprising amembrane-active fragment of gramicidin S or an E-alkylene isosterethereof conjugated to a cargo.
 96. The compound of claim 95, in whichthe gramicidin S fragment or E-alkylene isostere thereof comprises aβ-turn.
 97. The compound of claim 95, in which the gramicidin S fragmentor E-alkylene isostere thereof has the sequence Leu-^(D)Phe-Pro-Val-Orn.